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\begin{document}

\title{Human metapneumovirus in hospitalized children with acute respiratory
tract infections in Beijing, China}



\author[1]{Shanshan Cong}%
\author[2]{Chao Wang}%
\author[3]{Tianli  Wei}%
\author[4]{Zhiping Xie}%
\author[4]{Yi-man Huang}%
\author[2]{Jingjing Tan}%
\author[2]{Ai-jun Chen}%
\author[4]{Fenlian  Ma}%
\author[5]{Li-shu Zheng}%
\affil[1]{National Institute for Viral Disease Control and Prevention Beijing, CN}%
\affil[2]{National Institute for Viral Disease Control and Prevention, China CDC}%
\affil[3]{Beijing Friendship Hospital, Capital Medical University}%
\affil[4]{National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China}%
\affil[5]{National Institute for Viral Disease Control and Prevention}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Background This study aims to described the epidemiology and genotypic
diversity of Human metapneumovirus (HMPV) and the impact of SARS-CoV-2
on the prevalence of HMPV in hospitalized children with Acute
respiratory tract infections (ARTIs) in Beijing, China. Methods From
April 2018 to March 2019 and from September 2020 to August 2021,
nasopharyngeal aspirates (NPAs) from hospitalized children with ARTIs in
Beijing were collected and subjected to real-time polymerase chain
reaction tests for HMPV. Then genotyping, detection of 15 common
respiratory viruses and clinical characteristics were analyzed on HMPV
positive samples. Results 7.9\% (124/1572) enrolled paediatric patients
were identified as having HMPV infection, and the majority of children
under the age of 5 (78.2\%, 92/124), From April 2018 to March 2019. The
detection rate of HMPV in spring and winter is significantly higher than
that in summer and autumn. The co-infection rate were 37.1\% (46/124),
the most common co-infected virus were parainfluenza virus type 3
(HPIV-3). The main diagnosis of HMPV infection was pneumonia
(92.7\%,115/124), most patient have cough and fever. Of 78 HMPV-positive
specimens, A2b (82.1\%,64/78) were the main epidemic subtypes. .During
the COVID-19 outbreak, Among 232 samples, only 4 cases were
HMPV-positive. After statistical test, the detection rate of HMPV during
the COVID-19 pandemic has decreased significantly compared with that
before the epidemic (p=0.001). Conclusions HMPV is an important cause of
ARTIs in children under 5 years old. Under the prevention and control of
the COVID-19 pandemic, the HMPV infection of hospitalized children with
ARTIs has decreased significantly.%
\end{abstract}%



\sloppy


\textbf{Human metapneumovirus in hospitalized children}

\textbf{with acute respiratory tract infections in Beijing, China}

\textbf{Running title:} Epidemiology and Genotypic Diversity of HMPV

Shanshan Cong\textsuperscript{1,2}, Chao Wang\textsuperscript{1}, Tianli
Wei\textsuperscript{3}, Zhiping Xie\textsuperscript{1}, Yiman
Huang\textsuperscript{1}, Jingjing Tan\textsuperscript{1,2}, Aijun
Chen\textsuperscript{1}, Fenlian Ma\textsuperscript{1*} and Lishu
Zheng\textsuperscript{1, 4*}

\textsuperscript{1} NHC Key Laboratory of Medical Virology and Viral
Diseases, National Institute for Viral Disease Control and Prevention,
China CDC, Beijing, China

\textsuperscript{2}Department of Toxicology, School of Public Health,
China Medical University, Shenyang, P.R.China

\textsuperscript{3} Department of Pediatrics, Beijing Friendship
Hospital, Capital Medical University, Beijing, China

\textsuperscript{4} Center for Biosafety Mega-Science, Chinese Academy
of Sciences, Beijing, China

\textbf{*Correspondence:} Fenlian Ma and Lishu Zheng, National Institute
for Viral Disease Control and Prevention, China CDC, 100 Ying-Xin St.,
Xi-Cheng District, Beijing 100052, China. E-mails: mfl0729@126.com (F.
Ma) and zhengls@ivdc.chinacdc.cn (L. Zheng)

\textbf{Acknowledgements}

We thank the Department of Pediatrics, Beijing Friendship Hospital
(Capital Medical University) for providing the NPA specimens.

\textbf{Conflict of Interest statements}

The authors declare that there are no conflicts of interest.

\textbf{Abstract} :

\textbf{Background} This study aims to described the epidemiology and
genotypic diversity of Human metapneumovirus (HMPV) and the impact of
SARS-CoV-2 on the prevalence of HMPV in hospitalized children with Acute
respiratory tract infections (ARTIs) in Beijing, China.

\textbf{Methods} From April 2018 to March 2019 and from September 2020
to August 2021, nasopharyngeal aspirates (NPAs) from hospitalized
children with ARTIs in Beijing were collected and subjected to real-time
polymerase chain reaction tests for HMPV. Then genotyping, detection of
15 common respiratory viruses and clinical characteristics were
analyzed~on HMPV positive samples.

\textbf{Results} 7.9\% (124/1572) enrolled paediatric patients were
identified as having HMPV infection, and the majority of children under
the age of 5 (78.2\%, 92/124), From April 2018 to March 2019. The
detection rate of HMPV in spring and winter is significantly higher than
that in summer and autumn. The co-infection rate were 37.1\% (46/124),
the most common co-infected virus were parainfluenza virus type 3
(HPIV-3). The main diagnosis of HMPV infection was pneumonia
(92.7\%,115/124), most patient have cough and fever. Of 78 HMPV-positive
specimens, A2b (82.1\%,64/78) were the main epidemic subtypes.
Hospitalized children with HMPV genotype A infection had a higher viral
load compared to genotype B. During the COVID-19 outbreak, Among 232
samples, only 4 cases were HMPV-positive. After statistical test, the
detection rate of HMPV during the COVID-19 pandemic has decreased
significantly compared with that before the epidemic (\emph{p} =0.001).

\textbf{Conclusions} HMPV is an important cause of ARTIs in children
under 5 years old. The epidemic peak is generally in winter and spring,
and the A2b subtype is the most common. However, under the prevention
and control of the COVID-19 pandemic, the HMPV infection of hospitalized
children with ARTIs has decreased significantly.

\textbf{Keywords} Acute respiratory tract infections, Human
metapneumovirus , Epidemiology

\section*{1 Introduction}

{\label{introduction}}

Acute respiratory tract infections (ARTIs) remain one of the most common
major public health threats\textsuperscript{1}. According to the data of
the main causes of death in the world released by the World Health
Organization, Lower Respiratory Tract Infections (LRTIs) ranks fourth in
the world and ranks first in low-income countries\textsuperscript{2}.
Viral infections are the most frequent cause of
ARTIs\textsuperscript{3}. Since HMPV was first discovered in 2001 in the
Netherland and has been detected in other countries, it has been
determined to be one of the main pathogens of ARTIs in children,
immunocompromised people and the elderly\textsuperscript{4,5}. As a
member of the Metapneumovirus genus of the Pneumoviridae
family\textsuperscript{6}, HMPV genome is a negative-sense
single-stranded RNA molecule, 13Kb long, composed of eight genes
encoding nine proteins: 3'-N, P , M, F, M2-1/M2-2, SH, G,
L-5'\textsuperscript{7}. Based upon the sequence variability of the
attachment (G) and fusion (F) surface glycoproteins, HMPV can be divided
into four subtypes (A1, A2, B1 and B2). The A2 subgroup is the most
genetically heterogeneous of the four subgroups and some studies have
suggested its further sub-division into A2a, A2b1 and A2b2(A2c)
sub-lineages based on sequence data\textsuperscript{7-9}. It is well
known that these 2 main subgroups are prevalent around the world and
that major HMPV subtypes switch every year\textsuperscript{10-12}.
However, it is questionable whether there is an association between
genotype and disease severity.

Since late 2019, severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) was discovered in Wuhan, China, it has caused a huge
economic and health burden worldwide. To prevent the spread of the
virus, large-scale non-pharmaceutical interventions have been
implemented in China, including working from home, online teaching,
mandatory wearing of masks in public places, social distancing, and hand
hygiene. Reductions in influenza and respiratory~syncytial~virus cases
under these interventions have been reported in many countries, yet few
studies have examined changes in HMPV cases during
outbreaks\textsuperscript{13-17}. The purpose of this study was to
evaluate the epidemiological, clinical, and molecular characteristics of
HMPV infections occurring among hospitalized children with ARTIs in
China from April 2018--March 2019. In addition, this study explored the
impact of the COVID-19 pandemic on HMPV infection.

\section*{2 Materials and methods}

{\label{materials-and-methods}}

\subsection*{2.1 specimen collection}

{\label{specimen-collection}}

From April 2018 to March 2019, 1572 NPAs samples were collected from
hospitalized children (aged \textless{} 14 years) with ARTIs in Beijing
Friendship Hospital, and 233 samples were collected from September 2020
to August 2021. The study protocol was approved by the Ethical Committee
of Beijing Friendship Hospital (number: IVDC2018-012). Informed consent
has been received from the parent or guardian of the children
participating in the study\textbf{.} ARTIs were defined as at least two
of the following clinical manifestations during the previous week:
fever, cough, nasal obstruction, expectoration, sneeze and dyspnoea.
Patients with pneumonia diagnosed by chest radiography were also
included, even if they did not exhibit the clinical features described
above\textsuperscript{18}. Collected samples were stored in virus
preservation solution and placed at -80 for further processing.

\subsection*{2.2 Detection of HMPV}

{\label{detection-of-hmpv}}

Total viral nucleic acids were extracted from 200ul specimen and eluted
with 60ul water by using a QIAamp MinElute Kit (Qiagen, Germany)
according to the manufacturer's instructions. HMPV detection was
performed by using a quantitative real-time reverse transcription PCR
(qRT-PCR) assay with a One-step RT-PCR Kit (Ambion, USA). The HMPV
qRT-PCR primer set was used as previously described, designed based on
the conversed fragment of N gene \textsuperscript{19}: HMPV forward
primer(5'- TTAARTTACAAAAAAACATGGGAC -3'), reverse primer (5'-
AAAGAATATCTTTTCCTTCAGGG -3'), and probe (5'-FAM-
AATTACTCATAATCATTTTGACTG -3'-TAMRA). Each 25 \selectlanguage{greek}μ\selectlanguage{english}L reaction mixture
comprised 12.5 \selectlanguage{greek}μ\selectlanguage{english}L of RT-PCR 2\selectlanguage{ngerman}×Buffer, 1 \selectlanguage{greek}μ\selectlanguage{english}L of each primer (10 pM), 0.3
\selectlanguage{greek}μ\selectlanguage{english}L of probe (10pM) , 7.2 \selectlanguage{greek}μ\selectlanguage{english}L of Nuclease-Free water, 1\selectlanguage{greek}μ\selectlanguage{english}L of 25\selectlanguage{ngerman}×RT-PCR
Enzyme Mix and 2.0 \selectlanguage{greek}μ\selectlanguage{english}L of the nucleic acid components extracted from each
sample. The qRT-PCR cycling program was as follows: 45 \selectlanguage{ngerman}°C for 10 min, 95
°C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 45
s. Samples with a cycle threshold (Ct) \textless{} 38 were considered
positive. Measure of viral load based on a standard curve that has been
established in our laboratory\textsuperscript{20}.

\selectlanguage{english}\subsection*{2.3 Detection of viral co-infection in HMPV-positive
specimens}

{\label{detection-of-viral-coinfection-in-hmpvpositive-specimens}}

The HMPV-positive specimens were subsequently screened for 15 common
respiratory viruses.: influenza virus types A, B, and C (IFV A/B/C),
parainfluenza virus types 1--4 (HPIV), human coronaviruses
HKU1/229E/OC43/NL63 (HCoV), respiratory syncytial virus (RSV), human
rhinovirus (HRV), adenovirus (ADV), and human bocavirus (HBoV). All
viruses were detected by real-time polymerase chain
reaction\textsuperscript{21,22}, DNA viruses were tested using TaqMan\selectlanguage{english}
Gene Expression Master Mix (Thermo Fisher, USA), RNA viruses were tested
using AgPath-ID\selectlanguage{english} One-Step RT-PCR Kit (Ambion, USA) in accordance the
corresponding manufacture's protocols.

\subsection*{2.4 HMPV genotyping}

{\label{hmpv-genotyping}}

The viral RNA of HMPV positive samples was reverse transcribed into cDNA
using a SuperScript\selectlanguage{english} IV First-Strand Synthesis System (Invitrogen, USA)
in accordance with the manufacture's protocols. The conserved sequence
of F gene (610bp) was amplified using a nested PCR for genotyping as
previously described\textsuperscript{23}. The outer primers used were
forward 5\selectlanguage{english}'- CAATGCAGGTATAACACCAGCAATATC -3\selectlanguage{english}' and reverse 5\selectlanguage{english}'-
GCAACAATTGAACTGATCTTCAGGAAAC -3\selectlanguage{english}', and the internal primers were forward
5\selectlanguage{english}'- ACATGCCAACATCTGCAGGACAAATAAAAC -3\selectlanguage{english}' and reverse 5\selectlanguage{english}'-
ACATGCTGTTCACCTTCAACTTTGC -3\selectlanguage{english}'. Nested PCR was conducted in 25 \selectlanguage{greek}μ\selectlanguage{english}L volume
comprising 2.5 \selectlanguage{greek}μ\selectlanguage{english}L of 1 0 \selectlanguage{ngerman}× Ex Taq buffer, 2.0 \selectlanguage{greek}μ\selectlanguage{english}L of dNTP Mix, 1.0 \selectlanguage{greek}μ\selectlanguage{english}L(10
pM) of each primer, 0.4 \selectlanguage{greek}μ\selectlanguage{english}L of Ex Taq DNA polymerase, 2.0 \selectlanguage{greek}μ\selectlanguage{english}L of viral
nucleic acid extract or first nested-PCR product, and 16.1 \selectlanguage{greek}μ\selectlanguage{english}L of
double-distilled water. The Nested-PCR products were sequenced at Ruibio
Co., Ltd (Beijing, China), and a Maximum Likelihood (ML) tree was
constructed by the Tamura-Nei model in MEGA 7.0 with 1000 bootstrap
replicates. Samples that failed to be amplified were classified as
untyped.

\subsection*{2.5 Statistical analysis}

{\label{statistical-analysis}}

SPSS 26.0 software was used for data analysis, and Categorical data were
tested using \selectlanguage{greek}χ\selectlanguage{english}2 test and Fisher's exact test. Wilcoxon's test and
independent-samples t-test were used to analyze continuous variables.
Two-sided P-value \textless{} 0.05 was considered statistically
significant.

\section*{3 Results}

{\label{results}}

\subsection*{3.1 HMPV epidemiology}

{\label{hmpv-epidemiology}}

From April 2018 to March 2019, there were 900 males and 672 females
among the 1572 samples (sex ratio: 1.34:1), and the age range was from 1
day to 14 years of age with a median age of 3 years. The detection rate
of HMPV was 7.9\% (124/1572), of which 78 were male and 46 were female,
with no significant gender difference (\emph{p} =0.185). As shown in
Table 1, HMPV was detected among hospitalized children (\textless{} 14
years) with ARTIs at Beijing Friendship Hospital between April 2018 and
March 2019, and 78.2\% (97/124) of children under 5 years old. The
seasonal distribution of HMPV cases in hospitalized children from April
2018 to March 2019 is shown in Figure. 1, and HMPV was detected
throughout the year. 34.7\%(43/124)cases of HMPV infection were
detected in spring, 13.4\%(17/124)in summer, 13.4\%(17/124)in autumn
and 37.9\%(47/124)in winter, and there were significant differences in
HMPV detection rates in different seasons (\emph{p} = 0.000). From
September 2020 to August 2021, A total of 232 hospitalized children with
ARTIs were enrolled in this study, among which 134 (57.8\%) were male
and 98(42.2\%) were female (sex ratio: 1.34:1). The detection rate of
HMPV was 1.7\% (4/232), which was significantly lower than that of the
previous year, and the 3 positive cases were children under 5 years old.

\subsection*{3.2 Detection of viral co-infection in HMPV-positive
specimens}

{\label{detection-of-viral-coinfection-in-hmpvpositive-specimens-1}}

A total of 124 HMPV-positive samples were detected during the period
from April 2018--March 2019, of which 62.9\% (78/124) of HMPV
single-infected samples and 37.1\% (46/124) of HMPV co-infected samples
with other respiratory viruses. Among the 46 co-infected cases, there
were 32 males and 14 females (sex ratio:2.29:1), with no statistical
significance for gender (\emph{p} = 0.238). As shown in Table 2, the
most common mixed infection virus was HPIV3 (32.6\%,15/46), and the
mixed infection rates with HRV, ADV, FLV-A, and RSV were 19.6\% (9/46),
17.4\% (8/46), 17.4\% (8/46), and 10.9\% (5/46), respectively. The viral
load of the 124 HMPV positive cases ranged from 14 copies/mL to
4.6\selectlanguage{ngerman}×10\textsuperscript{6} copies/mL NPA, with no statistical difference
in viral load between HMPV mono-infection and coinfection. From
September 2020 to August 2021, among the NPAs samples from 232
hospitalized children with ARTIs, 4 were HMPV-infected, with viral loads
ranging from 19 copies/mL to 3.16×10\textsuperscript{3} copies/mL NPA. 2
cases were combined with other respiratory virus infections, which were
mixed with HBOV and HRV respectively.

\subsection*{3.3 Clinical characteristics of HMPV
infections}

{\label{clinical-characteristics-of-hmpv-infections}}

From April 2018 to March 2019, among the 124 children with HMPV
infection, 115 (92.7\%) were diagnosed with pneumonia, 8 (6.5\%) with
bronchitis, and 1 with upper respiratory tract infection (0.8\%). The
main clinical symptoms of children with HMPV infection were cough
(95.2\%, 118/124) and fever (91.9\%, 114/124), and other symptoms
included rhinorrhoea (53.2\%, 66/124), nasal obstruction (29.8\%,
37/124) 124), sneeze (15.3\%, 19/124) and Shiver (8.1\%, 10/124). There
was no statistical difference in the above clinical symptoms between
children with HMPV single infection and mixed infection, as shown in
Table 3. The hospitalization time of HMPV-infected children was 2-14
days, and the average hospitalization time was 6.35 days. A total of 90
children were discharged within 7 days (72.6\%, 90/124). Statistics on
the number of children who were hospitalized for more than 7 days showed
no significant difference between HMPV combined with other viral
infections and HMPV single infection (\emph{p} =0.320).

\subsection*{3.4 HMPV genotyping and phylogenetic
analysis}

{\label{hmpv-genotyping-and-phylogenetic-analysis}}

Nested PCR was used to amplify the conserved sequence (610bp) of the F
gene in 128 HMPV-positive samples, and the target fragment was amplified
from 78 (63.0\%, 78/124) HMPV-positive samples from April 2018 to March
2019. Phylogenetic analysis showed that 64 strains (82.1\%, 64/78)
belonged to A2b subtype, 9 strains (11.5\%, 9/78) B1 subtype and 5
strains (6.4\%, 5/78) B2 subtype, A1 and A2a subtype was not found in
Figure2. As shown in Figure3, the viral load of A2b subtype samples was
significantly higher than that of B type samples (\emph{p} =0.009), and
there was a statistically significant difference in the hospitalization
time \textgreater{}7 days between the two subtypes (\emph{p} =0.031).
From September 2020 to August 2021, among the 4 HMPV-infected children,
only 1 case was positive for F gene amplification, and was identified as
B1 subtype by phylogenetic analysis.

\section*{4 Discussion}

{\label{discussion}}

Since its discovery in 2001, HMPV has been considered an important cause
of ARTIs. Some studies have shown that the prevalence of HMPV is
different in different regions and years. The detection rate of HMPV in
Chinese hospitalized children with ARTIs is about
2\%-18.2\%\textsuperscript{18,24,25}, and the detection rate of HMPV in
ARTIs inpatients in other countries is
1.2\%-20.3\%\textsuperscript{12,26,27}. In this study, we mainly
explored the molecular epidemiology and clinical characteristics of HMPV
infection in hospitalized children with ARTIs in Beijing from April 2018
to March 2019 and September 2020 to August 2021, and the impact of the
COVID-19 epidemic on HMPV infection. The detection rate of HMPV from
April 2018 to March 2019 was 7.9\%, and the detection rate of HMPV from
September 2020 to August 2021 was 1.7\%. A previous study in our
laboratory showed that the detection rate of HMPV in hospitalized
children from April 2017 to March 2018 was 4.1\%\textsuperscript{25},
which indicated that the detection rate of HMPV in hospitalized children
with ARTIs in Beijing from April 2018 to March 2019 was higher than the
previous year (\emph{p} =0.000), while the detection rate of HMPV
decreased significantly during the period of the COVID-19 epidemic. It
shows that the detection rate of HMPV in hospitalized children with
ARTIs has been significantly reduced under the strict prevention and
control of the COVID-19 epidemic in China(\emph{p} =0.001). Mandy
Jongbloed et al. reported that despite a 324\% increase in HMPV testing
during the COVID-19 outbreak in Europe, there was no increase in HMPV
incidence\textsuperscript{28}. Since the outbreak of the COVID-19,
various countries have adopted different prevention and control
strategies. A series of measures such as wearing masks, frequent hand
washing and disinfection, delaying the start of school, and maintaining
social distance have cut off the transmission of respiratory viruses and
reduced HMPV infection. During the COVID-19 outbreak, the number of
hospitalized children with ARTIs decreased significantly, and the number
of samples collected from September 2020 to August 2021 decreased
accordingly. Due to the small number of HMPV-positive samples, the virus
mixed infection and clinical characteristics have not been counted yet.

From April 2018 to March 2019, 78.2\% of HMPV infections occurred in
hospitalized children aged \selectlanguage{english}[?]5 years, and 9.1\% (16/176) occurred in
children aged 3-4 years; There was no significant difference in HMPV
detection rate between male and female children(\emph{p} =0.185), which
was consistent with the studies in China and
Argentina\textsuperscript{25,27} . HMPV infection had typical seasonal
distribution characteristics, with the detection rate in winter and
spring significantly higher than that in summer and autumn (\emph{P}
=0.000), and the prevalence period of HMPV was concentrated from
December to May of the following year, which was consistent with the
results of a study in Guangzhou, China\textsuperscript{23}. Research
shows that HMPV epidemics are typical in early spring, while small peaks
in summer appear to be related to local weather
conditions\textsuperscript{22,27}. In ARTIs cases, HMPV is often
co-infected with other respiratory viruses, such as ADV, RSV, HRV and
HPIV\textsuperscript{25,29,30}. In this study, from April 2018 to March
2019, the proportion of HMPV-positive samples mixed with other viruses
was 37.1\%, of which HPIV3 had the highest mixed infection rate of
32.6\%, which was similar to the findings of Fathima
S\textsuperscript{27}, which may related to the prevalence of HPIV3 in
that year. Some studies have concluded that co-infection of HMPV with
other respiratory viruses can aggravate clinical symptoms and prolong
hospitalization\textsuperscript{18,32}. However, in this study, there
were no statistically significant differences in viral load, length of
hospital stay, cough, fever, nasal obstruction, rhinorrhoea and other
clinical symptoms between children with HMPV single infection and mixed
infection, which is consistent with the results of previous research in
our laboratory\textsuperscript{25}. In addition, the relationship
between HMPV viral load and clinical symptoms remains
unclear\textsuperscript{25,33,34}, and no association between clinical
symptoms and HMPV viral load was found in the study.

There are five gene subtypes in HMPV, so it is of great significance to
explore the relationship between genetic diversity of HMPV and clinical
characteristics. In this study, for HMPV samples from April 2018 to
March 2019, 78 NPAs samples were successfully amplified by nested PCR
amplification of the conserved fragment of the F gene. The phylogenetic
analysis showed that 82.1\% (64/78) of the prevalent strains belonged to
A2b subtype, 11.5\% belonged to B1 subtype and 6.4\% belonged to B2
subtype, and no A1 and A2a subtypes were found. We found that the viral
load of HMPV A2b genotype samples was significantly higher than that of
B samples (\emph{p} =0.009). In addition, there was a statistically
significant difference between A2b subtype and B subtype in the number
of children with hospitalization time \textgreater{} 7 days, and the
hospitalization time of A2b subtype was longer than that of B1 subtype
(\emph{p} =0.030), which suggests that we should pay more attention to
A2b subtype in pediatric clinic. From September 2020 to August 2021,
only one HMPV case was identified by phylogenetic analysis as subtype B1
infection. From April 2017 to March 2018, the detection rate of B1
subtype (54.5\%) were highest, followed by the A2b subtype (40.9\%) in
the previous study of our laboratory, and no HMPV A1 and A2a subtypes
were found\textsuperscript{25}. However, in this study, the A2b subtype
was the predominant (82.1\%), indicating that the main circulating
strain of HMPV infection in Beijing in 2017-2019 switched from B1
subtype to A2b subtype. The study by Matsuzaki Y showed that laryngitis
is more common in children with HMPV subtype B1 infection, while
wheezing is more common in children infected with HMPV subtypes B1 and
B2 than in children with subtype A2 infection\textsuperscript{35}. Many
data suggest that HMPV genotypes are associated with disease severity or
clinical manifestations \textsuperscript{36,37}, but some studies have
inconsistent conclusions \textsuperscript{24,38}, so it is still
controversial whether HMPV genotypes are associated with clinical
features.

This study was limited by the large decrease in hospitalized children
with ARTIs during the COVID-19 outbreak. From September 2020 to August
2021, there were only 4 HMPV positive children of 232 hospitalized
children with ARTIs, therefore a detailed epidemiological analysis of
HMPV cases during this period was not possible. We will continue to
collect respiratory samples from hospitalized children with ARTIs in the
subsequent studies, and make a detailed analysis of the impact of
SARS-CoV-2 on the prevalence of HMPV.

\textbf{Data availability}

Condensed anonymized data are available from the corresponding author on
reasonable request.

\textbf{Funding}

This work was supported by the Key Technologies R\&D Program of the
National Ministry of Science, China (,2018ZX10713002). The funding body
played no role in the design of the study and collection, analysis, and
interpretation of data and in writing the manuscript.

\textbf{Conflict of Interest statements}

The authors declare that there are no conflicts of interest.

\textbf{Ethical approval}

The project was approved by the Ethical Committee of National Institute
for Viral Disease Control and Prevention, China CDC, and the committee's
reference number is IVDC2018-012. All individuals (or their parents) in
the study population were informed about the current study and a written
informed consent was obtained from each subject.

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2013;46(5):352-7. doi:10.1016/j.jmii.2012.07.007

\textbf{TABLE1} HMPV infections in children of different ages and gender
with ARTIs\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Variable\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Number of patients\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Number of HMPV positive children\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Prevalence of HMPV (\%)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Gender\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Male\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
900\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
78\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
8.7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Female\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
672\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
46\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6.8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Age(years)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
[?]1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
420\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
29\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6.9\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textgreater{}1to[?]2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
204\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
8.8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textgreater{}2to[?]3\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
328\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
29\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
8.8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textgreater{}3to[?]4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
176\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
9.1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textgreater{}4to[?]5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
91\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
5.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textgreater{}5to[?]7\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
145\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
11\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textgreater{}7to[?]14\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
208\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7.7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Total\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1572\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
124\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7.9\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE2} Co-detection of HMPV and other respiratory viruses\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Groups\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
Virus composition\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
No. (\%)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
2018.4-2019.3 2 Virus (n=37)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
HMPV+HPIV3\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
13(28.3)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+RV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
6(13.0)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+IFV-A\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
6(13.0)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+RSV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
4(8.7)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+ADV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
4(8.7)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+ IFV-B\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+ IFV-C\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+HCoV-OC43\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+HPIV4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
3 Virus (n=7)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+ADV+HBOV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+ADV+IFV-C\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+HBOV+RV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+HBOV+RSV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+RV+HPIV4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+IFV-A+IFV-B\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+ HCoV-OC43+HPIV3\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
4 Virus (n=2)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+ADV+RV+HPIV3\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+ADV+HBOV+IFVA\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
2020.9-2021.8 2 Virus(n=2)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
HMPV+RV\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
1(50)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
HMPV+HBOV\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1(50)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE3} Clinical features among HMPV-positive hospitalized
children in 2018-2019\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Clinical characteristics\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Single infection NO. (\%) (n = 78)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Co-infection NO. (\%) (n = 46)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Total NO. (\%) (n = 124)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
p\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Fever ([?] 38)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
71(91)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
43(95.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
114(91.9)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.886\textsuperscript{b}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Convulsion\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2(2.6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1(2.2)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3(2.4)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.000\textsuperscript{b}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Shiver\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6(7.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4(8.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10(8.1)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.000\textsuperscript{b}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Cough\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
74(94.9)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
44(95.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
118(95.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.000\textsuperscript{b}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Nasal obstruction\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
21(26.9)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
16(34.8)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
37(29.8)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.355\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Rhinorrhea\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
44(56.4)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
22(47.8)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
66(53.2)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.355\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Sneeze\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12(15.4)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7(15.2)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
19(15.3)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.980\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Vomit\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4(5.1)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4(8.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
8(6.5)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.687\textsuperscript{b}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Diarrhea\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4(5.1)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4(3.2)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.296\textsuperscript{c}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Hospitalization\textgreater{}7d\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
19(24.4)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
15(32.6)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
34(27.4)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.320\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Note: a \selectlanguage{greek}χ\selectlanguage{english}2-test; b Continuity correction; c Fisher's exact test.

\textbf{Figure legends}

\textbf{FIGURE 1} Seasonal distribution of HMPV in children with ARTIs
from April 2018 to March 2019

\textbf{FIGURE 2} Phylogenetic relationships of the strains detected in
HMPV-infected children.

The reference strain is marked with a ^, and the strains analysed in
this study are marked with a ^.

\textbf{FIGURE 3} HMPV viral loads in NPA from children with ARTIs with
different genotype from April 2018 to March 2019\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/FIGURE1/FIGURE1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/FIGURE2/FIGURE2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/FIGURE3/FIGURE3}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

